Nanochannel arrays and their preparation and use for high throughput macromolecular analysis

ABSTRACT

Nanochannel arrays that enable high-throughput macromolecular analysis are disclosed. Also disclosed are methods of preparing nanochannel arrays and nanofluidic chips. Methods of analyzing macromolecules, such as entire strands of genomic DNA, are also disclosed, as well as systems for carrying out these methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/223,589, which is a continuation application of U.S. patentapplication Ser. No. 14/081,322, filed Nov. 15, 2013, which is acontinuation application of U.S. patent application Ser. No. 12/261,406,filed Oct. 30, 2008, now issued as U.S. Pat. No. 8,652,828, which is adivisional application of U.S. patent application Ser. No. 10/484,293,filed Jan. 20, 2004, now issued as U.S. Pat. No. 7,670,770, which is theU.S. national phase application filed under 35 U.S.C. § 371 claimingbenefit to International Patent Application No. PCT/US02/23610, filedJul. 25, 2002, which is entitled to priority under 35 U.S.C. § 119(e) toU.S. Provisional application No. 60/307,668, filed Jul. 25, 2001, eachof which are incorporated herein by reference in their entireties.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under DARPA Grant NumberMDA972-00-1-0031 awarded by the Defense Advanced Research ProjectsAgency. The government has certain rights in the invention.

BACKGROUND

The present invention relates to a nanochannel array. The presentinvention also relates to a method of preparing nanochannel arrays. Thepresent invention also relates to nanofluidic chips containingnanochannel arrays. The present invention also relates to a systemsuitable for high throughput analysis of macromolecules. The presentinvention also relates to a method of analyzing at least onemacromolecule by using a nanochannel array.

In the newly emerging field of bionanotechnology, extremely smallnanofluidic structures, such as channels, need to be fabricated and usedas arrays for the manipulation and analysis of biomolecules such as DNAand proteins at single molecule resolution. In principle, the size ofthe cross sectional area of channels should be on the order of the crosssectional area of elongated biomolecules, i.e., on the order of 1 to 100square nanometers, to provide elongated (e.g., linear, unfolded)biomolecules that can be individually isolated, yet analyzedsimultaneously by the hundreds, thousands, or even millions. Likewise,it is also desirable that the length of the channels should be longenough to accommodate the longest of macromolecules, such as an entirechromosome, which can be on the order of 10 centimeters long (e.g.,chromosome 1 of the human genome having 250 million base pairs). Thepresent inventors and others have recently been concerned about suchproblems and their possible solutions, as reported in: O. Bakajin, etal., Anal. Chem. 73 (24), 6053 (2001), J. O. Tegenfeldt, et al., Phys.Rev. Lett. 86 (7), 1378 (2001), J. Han et al., Science 288, 1026 (2000),and S. R. Quake et al., Science 290 (5496), 1536 (2000).

It is important to efficiently and reliably construct arrays of manythousands, or even millions of channels in an array for the simultaneousisolation and analysis of up to thousands or millions of individualmacromolecules. Such large arrays of isolated macromolecules could, inprinciple, be analyzed with presently available two-dimensional areadetectors, such as charge-coupled devices (CCDs). Together withautomated data-processing collection and image analysis software, thesimultaneous characterization of up to thousands or millions ofmacromolecules would be an extremely powerful tool for macromolecularanalysis, such as population distribution analysis of macromolecularsize, chemical composition, and DNA sequencing.

Because individual macromolecules could in principle be isolated andanalyzed in a single channel, heterogeneity of a sample containing amultitude of macromolecules can be readily discerned. This would beparticularly useful for identifying single nucleotide polymorphisms(SNP) on a single chromosome. In contrast, traditional population basedassays require time-consuming DNA amplification methods to preparemultiple copies of a nucleic acid macromolecule to carry out SNPanalysis. If available, a chromosomal analysis system incorporatingnanochannel arrays could perform SNP analysis much more quickly than anymethod presently available.

Nanochannel arrays having the proper dimensions for carrying out thesimultaneous isolation and analysis of a multitude of elongatedmacromolecules have been heretofore unavailable. Accordingly, there isan urgent need to provide nanochannel arrays having at least three keydimensional qualities: (1) the channels should have a sufficiently smalldimension to elongate and isolate macromolecules; (2) the channelsshould have a sufficiently long dimension to permit the instantaneousobservation of the entire elongated macromolecule; and (3) a high numberof channels should be provided to permit the simultaneous observation ofa high number of macromolecules. In addition, it would be desirable forthe elongated and isolated macromolecules to remain indefinitely in sucha state at ambient conditions even after the field which is used totransport the macromolecules into the channels (e.g., electric field) isturned off. This feature would permit the macromolecules to be analyzedwith techniques that require times longer than the residence time of themacromolecule under the influence of the field. This feature would alsopermit the analysis of macromolecules without having to subject them toa field.

Methods for analyzing macromolecules (e.g., polymers) have beenpreviously disclosed, however none uses a nanochannel array having thethree key dimensional qualities as described supra. U.S. Pat. No.5,867,266 discloses a micro optical system having a plurality ofcoplanar micron-to-millimeter scale sample channels prepared usingphotolithography and an artificial gel material comprising amultiplicity of pillar structures in each micron-to-millimeter widesample channel. The large channel width makes this system unsuitable asa nanochannel array.

Likewise, methods for analyzing macromolecules (e.g., polymers) byisolating them in channels more narrow than this disclosed in U.S. Pat.No. 5,867,266, however none uses a nanochannel array having the threekey dimensional qualities as described supra. In WO 00/09757, several ofthe inventors of the present patent application disclose a system foroptically characterizing single polymers that are transported in astraightened form through a channel. In U.S. Pat. No. 6,355,420, asystem is disclosed for analyzing polymers that are transported in astraightened form through a plurality of (at least 50) channels. Whileboth of these disclosures are directed towards analysis of singlemacromolecules aligned in one or more channels, neither of thesedocuments discloses the simultaneous observation of a high number ofmacromolecules in a multitude of channels.

Thus, there remains the problem of providing suitable nanochannel arraysthat are useful in a variety of macromolecular analysis. Methods foranalyzing macromolecules (e.g., polymers) by isolating them in a narrowchannel have been previously disclosed, however none uses a nanochannelarray having the three key dimensional qualities as described supra,primarily because, until now, fabrication techniques for constructingsuch a nanochannel array were not available.

In creating ultra-small nanofluidic structures, e.g. for singlebiomolecule analysis, at least two problems need to be solved: reductionof size and creation of sealed fluidic channels. As reported by one ofthe present inventors, NIL is a parallel high-throughput technique thatmakes it possible to create nanometer-scale features over largesubstrate surface areas at low cost. (S. Y. Chou et al., Appl. Phys.Lett. 67 (21), 3114 (1995) and S. Y. Chou et al., Science 272, 85(1996)) Current sealing techniques such as wafer bonding (M. Stjernstromet al., J. Micromech. and Microeng. 8 (1), 33 (1998)), and softelastomer sealing (H. P. Chou et al., Proc. Nat. Acad. Sci. USA 96 (1),11 (1999), are suitable for relatively large planar surfaces and providean effective seal. Wafer bonding requires an absolutely defect free andflat surface, and elastomer sealing suffers from clogging due to softmaterial intrusion into the channels. Within extremely small confiningstructures, biological samples are also much more sensitive to issuessuch as hydrophobicity and the homogeneity of the material constructingthe fluidic structure.

Recently developed techniques using “place-holding” sacrificialmaterials such as polysilicon (S. W. Turner et al., J. Vac. Sci. andTechnol. B 16(6), 3835 (1998)) and polynorbornene (D. Bhusari et al., J.Microelectromech. Syst. 10 (3), 400 (2001)) have gained popularity tocreate sealed small hollow fluidic structures. However, steps needed inremoving the sacrificial materials such as heating the substrate up to200-400° C. or wet etching limits the use of certain materials anddownstream fabrication processes.

As provided herein, the present invention achieves the goal of providingnanochannel arrays suitable for performing high throughputmacromolecular analysis. Interferometric lithography (IL), nanoimprintlithography (NIL), and non-isotropic deposition techniques are used toprepare nanochannel arrays having hundreds of thousands to more than amillion enclosed channels having the desired key dimensions across thesurface of a silicon wafer substrate.

In one aspect of the present invention, there are provided nanochannelarrays including a surface having a plurality of channels in thematerial of the surface, said channels having a trench width of lessthan about 150 nanometers and a trench depth of less than 200nanometers; at least some of the channels being surmounted by sealingmaterial to render such channels at least substantially enclosed.

In a further aspect of the present invention, methods of preparingnanochannel arrays are disclosed, which include the steps of: providinga substrate having a surface; forming a plurality of channels in thematerial of the surface; and depositing a sealing material on theplurality of channels to surmount the plurality of channels to rendersuch channels at least substantially enclosed, the substantiallyenclosed channels having a trench width of less than 150 nanometers anda trench depth of less than 200 nanometers.

In another aspect of the invention, there are provided nanofluidic chipsincluding: a) nanochannel array, including: a substrate having asurface; a plurality of parallel channels in the material of thesurface, said channels having a trench width of less than about 150nanometers and a trench depth of less than 200 nanometers; at least someof the channels being surmounted by sealing material to render suchchannels at least substantially enclosed; at least some of the channelsare capable of admitting a fluid; b) at least one sample reservoir influid communication with at least one of the channels, said samplereservoir capable of releasing a fluid; and c) at least one wastereservoir in fluid communication with at least one of the channels, saidwaste reservoir capable of receiving a fluid.

In yet another embodiment of this invention, there are provided systemsfor carrying out analysis. In exemplary embodiments, these include: A) ananofluidic chip, including: a) nanochannel array, including: asubstrate having a surface, a plurality of parallel channels in thematerial of the surface, said channels having a trench width of lessthan about 150 nanometers and a trench depth of less than 200nanometers; at least one of the channels being surmounted by sealingmaterial to render such channels at least substantially enclosed; atleast one of the channels capable of admitting a fluid; and b) at leastone sample reservoir in fluid communication with at least one of thechannels, said sample reservoir capable of releasing a fluid; and B) adata processor.

In another embodiment, methods of analyzing at least one macromoleculeare described which, for example, include the steps of: providing ananofluidic chip, including: a) nanochannel array, including: a surfacehaving a plurality of parallel channels in the material of the surface,said channels having a trench width of less than about 150 nanometersand a trench depth of less than 200 nanometers; at least one of thechannels being surmounted by sealing material to render such channels atleast substantially enclosed; at least one of the channels capable ofadmitting a fluid; b) at least one sample reservoir in fluidcommunication with at least one of the channels, said sample reservoircapable of releasing a fluid containing at least one macromolecule;providing the at least one sample reservoir with at least one fluid,said fluid comprising at least one macromolecule; transporting the atleast one macromolecule into the at least one channel to elongate saidat least one macromolecule; detecting at least one signal transmittedfrom the at least one elongated macromolecule; and correlating thedetected signal to at least one property of the at least onmacromolecule.

Cartridges including a nanofluidic chip in accordance with thisinvention are also disclosed herein. Such cartridges are capable ofbeing inserted into, used with and removed from a system such as thoseshown herein. Cartridges useful with analytical systems other than thesystems of the present invention are also comprehended by thisinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a cross-section of a nanochannel array havingsubstantially enclosed channels.

FIG. 2 illustrates a cross-section of a nanochannel array havingcompletely enclosed channels and having sealing material deposited inthe channels.

FIG. 3 is a scanning electron micrograph of a nanochannel array havingparallel linear channels and open channel ends.

FIG. 4 illustrates a schematic of a process for depositing sealingmaterial into the channels.

FIG. 5 illustrates a nanofluidic chip.

FIG. 6a is a scanning electron micrograph of the substrate used inExample 4 prior to sealing with silicon dioxide.

FIG. 6b is a scanning electron micrograph of the nanochannel array ofExample 4 obtained after sealing the substrate with silicon dioxide.

FIG. 6c is a scanning electron micrograph (top view) of the nanochannelarray of Example 5.

FIG. 6d is a scanning electron micrograph (top view) of the nanochannelarray of Example 4.

FIG. 7a is a scanning electron micrograph of the substrate used inExample 1 prior to sealing with silicon dioxide.

FIG. 7b is a scanning electron micrograph of the nanochannel array ofExample 1 obtained after sealing the nanochannel array in 7 a withsilicon dioxide.

FIG. 7c is a scanning electron micrograph of the substrate used inExample 2 prior to sealing with silicon dioxide.

FIG. 7d is a scanning electron micrograph of the nanochannel array ofExample 2 obtained after sealing the nanochannel array of 7 c withsilicon dioxide.

FIG. 7e is a scanning electron micrograph of the substrate used inExample 3 prior to sputtering with silicon dioxide.

FIG. 7f is a scanning electron micrograph of the nanochannel array ofExample 3 obtained after sputtering the nanochannel array in 7 e withsilicon dioxide.

FIG. 8a illustrates a sealed channel having a nanoslit in an opaquelayer across the bottom of a channel.

FIG. 8b illustrates a sealed channel having a nanoslit in an opaquelayer across the sealing layer, which is oriented perpendicular to thelong axis of a nanochannel.

FIG. 9a shows scanning electron micrographs of the substrate (left andbottom) and a of the sealed nanochannel array (right) used in Example14.

FIG. 9b is the image obtained from the CCD of the 48.5 kb lambda phagegenome (shorter) and 168 kb T4 phage genome (longer) of Example 14.Inset: Plot of genome size versus macromolecular contour length.

FIG. 9c shows a nanochannel array simultaneously elongating, separating,and displaying a plurality of DNA macromolecular ranging in size from 10kb to 196 kb.

FIG. 10 illustrates a system for analyzing macromolecules using ananofluidic chip.

One aspect of the present invention encompasses a nanochannel arrayhaving a plurality of channels that are substantially enclosed. As shownin FIG. 1, the nanochannel array 100 has a surface 102 that contains aplurality of channels 104 in the material of the surface 106. Thechannels 104 have a wall 110, and a channel center 112. The distancebetween the wall surfaces 110 inside a channel 104 that areperpendicularly opposite to the channel center 112 is defined as thetrench width. The channels 104 are surmounted by a sealing material 108that renders the channels 104 at least substantially enclosed.

In one embodiment, the channels 104 will not be completely enclosed andwill typically have no sealing material 108 directly above the channelcenter 112, providing an opening in the sealing material to the channel.The opening may have a variety of shapes. The size of the opening isdefined as the minimum distance spanning the opening above the channelcenter 112. In such embodiments, the size of the opening is less thanthe trench width, and is typically less than ½ of the trench width, moretypically less than ⅓ of the trench width, and most typically less than¼ of the trench width. In other embodiments the channels can becompletely enclosed, having sealing material completely covering the topof the channel and having no opening in the sealing material. In certainembodiments of the present invention, sealing material 108 can extendover the walls 110 and the bottom of the channels 104, as shown in FIG.2. In such embodiments, the trench width is defined as the distance fromthe surfaces formed by the sealing material adjacent to the walls 114.

In the present invention, the trench width is typically less than 150nanometers, more typically less than 100 nanometers, and even moretypically less than: 75, 50, 25, and 15 nanometers. In certainembodiments the trench width is about 10 nanometers. In the presentinvention, the trench width is at least 2 nm, and typically at least 5nm.

In the present invention the channels are at least substantiallyenclosed. “At least substantially enclosed” means that the channels arecompletely enclosed or have an opening in the sealing material that issmaller than ½ the trench width, or have both completely enclosedchannels and openings.

Channels that are completely enclosed have a trench depth that isdefined as the distance between the surface of the solid material at thebottom of the channel below the channel center 112 to the sealingmaterial above the channel center 112. Embodiments in which the channelshaving an opening have a trench depth defined as the distance from thesurface of the solid material at the bottom of the channel below thechannel center to the position of the opening where the opening size ismeasured. If the opening has more than one position where a minimumdistance can be measured then the position of the opening is the onethat is closed to the bottom of the channel 104.

In the present invention, the trench depth is less than 200 nm. Incertain embodiments, the trench depth is typically less than 175 nm, andmore typically less than 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, and 25nm. In certain embodiments the trench depth is about 15 nm. In certainembodiments the trench depth is at least 2 nm, typically at least 5 nm,and more typically at least 10 nm.

In the present invention, the nanochannel arrays can be formed in asubstrate, such as a silicon wafer substrate, using a variety offabrication methods as described below. In one embodiment, thenanochannel array has a plurality of parallel linear channels across thesurface of substrate as illustrated by the scanning electron micrographin FIG. 3.

In certain embodiments, the nanochannel arrays have at least one end ofat least one of the channels can be in fluid communication with at leastone reservoir. In these embodiments, at least one channel is connecteddirectly with at least one reservoir. Alternatively, at least onechannel can be connected with at least one reservoir via aninterconnecting straight or curved microchannel (a channel having awidth, height, or both larger than about a micron), or a channel isconnected with at least one reservoir via an interconnecting nanopillaror micropillar array.

In certain embodiments, at least two ends of some of the channels are influid communication with at least one reservoir common to the channels.In these embodiments, at least two ends of some of the channels can beadjacent or not adjacent. These channels can be connected directly withat least one reservoir.

In certain embodiments, at least two channels can be connected with atleast one reservoir via a common interconnecting straight or curvedmicrochannel. Alternatively, at least two channels can be connected withat least one reservoir via a common interconnecting nanopillar ormicropillar array.

In certain embodiments of the present invention, the nanochannel arrayhas a plurality of channels that are in fluid communication with atleast one sample reservoir common to at least some of the channels. By“a plurality of channels” is meant more than two channels, typicallymore than 5, and even typically more than 10, 100, 1000, 10,000 and100,000 channels. In certain embodiments, one sample reservoir can beprovided for all of the channels that are on the substrate. In a certainembodiments, 100 mm diameter substrates can have about 500,000 parallellinear channels having a periodicity of 200 nm, the periodicity beingdefined as the distance between the middle of two adjacent channels.

In certain embodiments, the plurality of channels can be connecteddirectly with at least one reservoir. The connections can be a commoninterconnecting straight or curved microchannel. In other embodiments, aplurality of channels can be connected with at least one reservoir via acommon interconnecting nanopillar or micropillar array.

In certain embodiments of the present invention, the nanochannel arraycontains a plurality of channels that are in fluid communication with atleast one waste reservoir. Although the plurality of channels istypically connected directly with at least one waste reservoir, morethan one waste reservoir can also be provided. It should be appreciatedthat the waste reservoir can be used as a sample collection reservoir.Accordingly, multiple sample collection reservoirs can also be providedon nanochannel arrays. In these embodiments, a plurality of channels canbe connected with at least one waste reservoir via a commoninterconnecting straight or curved microchannel as described earlier.Likewise, a plurality of channels can be connected with at least onewaste reservoir via a common interconnecting nanopillar or micropillararray.

In certain embodiments of the present invention, the nanochannel arrayhas a plurality of channels that are substantially parallel, in whichthe plurality of channels are substantially in the plane of the surfaceof the substrate.

In certain embodiments of the present invention, the nanochannel arraycan contain linear channels. Linear channels are adjacent channels thatare substantially not interconnected.

In certain embodiments, the ends of the channels are capable ofadmitting a macromolecule in a fluid. By being capable of admittingmacromolecule means that the channels have at least one opening largeenough to permit the passage of a macromolecule. While a variety ofopenings are envisages, typically such openings can be located at theends of the channels or on the surface of the sealing material throughopenings in the sealing material. Openings in the sealing material canbe provided by subsequent modification of the nanochannel arrays asprovided below.

In certain embodiments of the present invention, the nanochannel arraycontains channels that are capable of transporting a macromoleculeacross their length. The nanochannel arrays can be fitted with a varietyof components to affect macromolecular transport, examples of whichinclude pressure or vacuum gradient drop across the channels,electroosmosis, and electrokinesis.

While not being bound to a particular theory, it is believed thatmacromolecules typically have a non-linear, three-dimensionalconformation in space (e.g., linear polymers have a random coilconformation in their natural state). Accordingly, it isthermodynamically unfavorable for macromolecules to spontaneouslyelongate and enter channels directly from the environment due to thelarge free energy needed to reduce entropy. For example, a 169 kilobaseT4 phage double stranded genomic DNA chains will form a Gaussian coil ofradius of gyration Rg=(Lp/6)^(1/4)=700 nm in free solution, where L isits calculated contour length and p is the persistence length of about50 nm.

In certain embodiments, the nanochannel array can contain channelscapable of transporting at least one macromolecule across the length ofchannels, in which the macromolecule is in an elongated form. Suchchannels can have openings large enough to permit the entrance of theends of the macromolecules. In certain embodiments, it is preferred thatsuch channels also have trench widths and trench depths narrow enough torestrict the movement of the macromolecules to primarily one directionalong the surface of the substrate. Preferably such channels are notinterconnected.

In certain preferred embodiments of the present invention, thenanochannel array is capable of transporting at least one biopolymeracross the length of said channels. In these embodiments, the geometryof the channels permits the biopolymers to enter and move along thechannels in at least one direction. Preferably, the channel surfaces aretreated with a non-sticking agent, as described later, for preventingthe adhesion of macromolecules, such as biopolymers, to the inside ofthe channels.

In other embodiments, it is preferred that the nanochannel arraycontains channels capable of transporting at least one unfoldedbiopolymer across the length of said channels. While not being bound bya particular theory, when the dimensions of the channels are apparentlylarger than the spatial conformation of the macromolecules, there is atleast a partial amount of elongation of the macromolecules in thechannels. When the dimensions of the channels are at the same order orbelow the persistence length of macromolecules, such as 50 nm for DNAthe macromolecules can be sufficiently elongated in an unfolded fashioninside the channels. When the dimensions of the channels fall in betweenthe above-mentioned two scenarios, macromolecules can be partiallyelongated in these channels. In this case, the macromolecules can befolded, tangled, or both folded and tangled. While it is envisaged thatany macromolecule can be transported in an unfolded fashion in thechannels of the nanochannel array of the present invention, a variety ofsuitable unfolded macromolecules include RNA, DNA, denatured unfoldedpeptide chains, self-assembled chemical polymers, and co-polymer chains,and other biopolymers, and combinations thereof.

In one preferred embodiment, the channel structures of the nanochannelarrays can be formed from linearly adjacent channel walls that span thesubstrate surface. In other embodiments, the channels can be formed frompillar structures, self-assembled polymer structures, stacked membranelayers, and nanobeads (particles inside the channels).

The surface material of the nanochannel arrays can be formed from almostany substrate material, such as a conductive material, a semiconductormaterial, or a non-conductive material. Examples of conductive materialsinclude metals such as aluminum, gold, silver, and chromium. Examples ofsemiconductive materials include doped silicon dioxide and galliumarsenide. Examples of non-conductive materials include fused silica,silicon dioxide, silicon nitride, glass, ceramics, and syntheticpolymers. The foregoing is exemplary only.

In the present invention, the surface of the nanochannel array istypically the surface of the substrate, such as the surface of a siliconwafer. Alternatively, the surface can be a film, such as one adjacentlysupported by a second substrate. Coating a material on to a secondsubstrate can form films. A suitable coating process includes vapordeposition of a material onto a wafer.

In certain embodiments of the present invention, the nanochannel arrayincludes at least one optically opaque material layer adjacent to thesealing material. The optically opaque material can be situated betweenthe surface material and the sealing layer, it can be situated insidethe channels, it can be situated on top of the sealing material, or acombination of these. While almost any opaque material that can bedeposited as a layer is possible in this embodiment, Aluminum ispreferred. For certain embodiments, it is desirable that the opaquelayer thicknesses are less than about 50 nm thick. For embodimentscontaining nanoslits useful for carrying out near-field imaging of thecontents of the channels, it is desirable to prepare slits smaller than50 nm that are etched through the deposited opaque layer but not throughthe transparent sealing material for maintaining the integrity of theadjacent (underneath) channel. Without being bound by a particulartheory, the optically opaque layer functions as a blocking mask forhigh-resolution near-field excitation. Without being bound to aparticular theory, aluminum is particularly preferred as an opaque layerbecause it has the highest known skin depth of any material at the givenwavelength of the excitation light source rendering the smallestthickness of the blocking layer, hence the shortest distance between theslit and the possible target molecules in channels.

In certain embodiments of the present invention, the nanochannel arrayhas at least one near field slit feature above at least one channel.Such slits should be fabricated as close (less than about 30 nm) to thechannel as possible without compromising the integrity of the adjacentsealed channels. The thin wall of sealing material between slit openingand channels could be created by FIB milling or controlled materialdeposition.

In a further preferred embodiment, the nanochannel array containssealing material adjacent to the channel bottom. Sealing material can beprovided in channel bottom by depositing a suitable sealing materialinto the channels prior to or simultaneously with enclosing thechannels.

The nanochannel arrays also preferentially have sealing materialadjacent to the channel wall material. In this embodiment, the sealingmaterial can reduce the trench width. This is particularly advantageousfor preparing nanochannel arrays from a variety of substrate surfacesthat contain channels wider than 150 nm in trench width and deeper than200 nm in trench depth. In this embodiment, and as described below,sealing material can be deposited into channels by a variety of methods.One suitable method is E-beam evaporation, which creates a point sourceof material. In E-beam evaporation, a substrate is typically far awayfrom the source compared to the size of the sample, and the angulardistribution of the depositing material is very narrow. To achieve anon-uniform deposition the substrate is tilted at a specific angle. Thechannel walls partially block the deposition of sealing material (like ashadow), and most of the material is be deposited on the channel wallsnear the upper portion of the channel wall. Beyond a critical depth nodeposition will occur as long as the substrate is tilted.

An alternative and preferred method to provide sealing material in thechannels is sputter deposition. In sputter deposition, the sealingmaterial is deposited at all angles, so the instant growth rate at anypoint on the surface depends on the percentage of the total target areawithin its line of sight, as is outlined in FIG. 4. Without being boundto a particular theory, sealing material from a large target source 130that is in close proximity to the substrate surface, can travel along avariety of trajectories (122, 124, 126, 127) and be deposited atdifferent positions in the channels. Sputtering is typically usedbecause of the divergent nature of the material beam, thus resulting inthe faster deposition of target material at the top part of the channelsinstead of at the bottom of the channels (i.e., surmounting thechannel). In time, the sealing material near the top of the channelseventually completely encloses the channel, which prevents furtherdeposition of sealing material into the channel. In one embodiment, theresultant sealing material in the channel results in a profile 128.Suitable sputtering systems are known in the art. A particularlysuitable sputtering system has a 200 mm SiO2 target source whichprovides high surface coverage and uniformity across 100 mm substrate.

The lengths of the channels of the nanochannel array can have a widerange. The lengths of the channels can also be the same or different inthe nanochannel array. For carrying out macromolecular analysis usingthe nanochannel arrays as provided below, it is desirable that thechannels are at least 1 millimeter (mm) longer. More typically, thelength of the channels is greater than 1 centimeter (cm), and evengreater than 5 cm, 15 cm, and 25 cm.

In another aspect of the present invention there is provided a method ofpreparing a nanochannel array, which includes the steps of forming aplurality of channels in the material of the surface of a substrate, anddepositing a sealing material to surmount the plurality of channels toprovide at least substantially enclosed channels. Substrates containinga plurality of channels preferably have a periodicity of 200 nanometersor less, which can be provided by interferometric lithography andnanoimprint lithography techniques, which are disclosed in U.S. Pat. No.5,772,905, the complete disclosure of which is incorporated by referenceherein. As described earlier, various types of materials can be used toprepare surfaces having a plurality of channels. Suitable substratesinclude semiconductors, dielectrics, polymers, self-assembled biofilm,membranes, metals, alloy, and ceramics.

The sealing material is preferably deposited to surmount the pluralityof channels to render such channels at least substantially enclosed, thesubstantially enclosed channels having a trench width of less than 150nanometers and a trench depth of less than 200 nanometers. By “surmount”is meant that the sealing material is preferentially deposited towardsthe top of the channels compared to the bottom of the channels,resulting in substantially enclosed channels, which is described aboveand in FIG. 4.

In certain embodiments of the present invention, the sealing materialcan be deposited using any of a variety of methods, including chemicalvapor deposition, spin coating, film lamination, and thermo-evaporation.Preferably, the sealing material is deposited using electron-beamevaporation or sputtering.

In certain embodiments of the present invention, sealing material isdeposited on substrate surfaces by a sputtering process at gas pressurestypically less than about 20 mTorr, more typically less than 10 mTorr,and even more typically less than 5 mTorr. Sputtering is a process ofdriving molecules off a source target surface (such as SiO₂) usingenergetic ionic bombardment. Atoms are knocked off from the sourcetarget and can be deposited on a variety of substrate surfaces, such aspatterned silicon wafers. While not being bound to a particular theory,it is believed that as the gas pressure is reduced, there are fewerparticles in the environment of the plasma sputtering chamber, whichresults in the depositing atoms to travel with fewer collisions beforereaching the substrate surface; hence, a more anisotropic and fasterdeposition. At higher gas pressure such as 30 mTorr, depositing atomscollide more frequently on their path to the substrate surface, hence amore divergent traveling angles and more isotropic and slowerdeposition. At lower gas pressure with more anisotropic and fastdeposition, more depositing atoms can reach the bottom and lower part ofthe sidewalls of the trenches, causing relatively faster deposition ofsealing material at the bottom and sidewalls comparing to the top of thetrenches, this subsequently leads to smaller channel (trench)dimensions.

The aspect ratio of the trenches being sealed also affects the geometryof the final sealed void space. The higher the depth to width ratio, theless sealing material will be deposited near the bottom of the trench.The lower the depth to width ratio, the smaller and narrower the channeldimensions.

In one embodiment of carrying out the method of the present invention,at least one reservoir is provided to be in fluid communication with atleast one end of at least one of the channels. Channels can befabricated on the substrate using nanoimprinting and interconnectingstructures of pillar arrays. Reservoirs can be defined usingphotolithography and subsequently pattern transferred to the substrateusing Reactive Ion etching (RIE), chemical etching or FIB millingdirectly to create reservoirs in fluid communication with the channels.Auxiliary structures, such as microchannels, for connecting thereservoirs to the channels can also be provided using these methods.Typical depth of the reservoirs and auxiliary structures is typically atleast several hundreds of nanometers, preferably at least severalmicrometers deep.

In certain embodiments, it is desirable to provide an additional sealingstep. A suitable additional sealing step includes application of aplanar surface substrate to the top of the sealing material.Alternatively, reservoirs can be formed on the sealing planar substrate.Auxiliary fluid communicating structures larger than about a micron canalso be formed to connect to larger sample reservoir. A variety ofschemes to connect reservoirs to the channels can be envisioned: atleast 2 reservoirs can be provided in fluid communication with at least2 separate channels; or at least 10 reservoirs are provided in fluidcommunication with at least 10 separate channels; or at least 96reservoirs are provided in fluid communication with at least 96 separatechannels; or at least 500 reservoirs are provided in fluid communicationwith at least 500 separate channels; or at least 5000 reservoirs areprovided in fluid communication with at least 100 separate channels; orcombinations thereof.

In a preferred embodiment of the present invention, the method ofpreparing the nanochannel arrays is carried out using linear channelarray substrates having a periodicity of less than 200 nm formed bynanoimprint lithography. In this embodiment, the linear channels have atrench width less than 100 nanometers and a trench depth less than 100nanometers. In this embodiment, at least a portion of the sealingmaterial is deposited using sputter deposition to provide sealingmaterial adjacent to the channel wall material to narrow the trenchwidth.

Varying the sealing material deposition parameters is also used tonarrow the trench width of the channels. The deposition parameters canbe varied to provide trench widths of typically less than 100nanometers. As more material is deposited, trench widths can be narrowedto less than 75 nanometers, and even less than: 50 nanometers, 25nanometers, and 15 nanometers. Trench widths of about 10 nm can also beprovided by the methods of the present invention. Typically, theresulting trench widths after deposition will be greater than 2 nm, andmore typically greater than 5 nanometers. Trench depths of less than175, 150, 125, 100, 75, 50, and 25 nm can also be provided by themethods of the present invention. Trench depths of about 15 nm can alsobe provided. Typically, the trench depths will be at least 5 nm, andmore typically at least 10 nm.

In another embodiment of the present invention, the method may alsoinclude the step of providing at least one near field slit feature aboveat least one channel. In this step, the sealing material is typicallytransparent, such as silicon dioxide, to permit spectroscopic detectionof fluorescently labeled macromolecules, such as DNA, inside thechannels. This permits the use of optical methods, such as near-fieldoptical imaging, to analyze macromolecules in the channels. Nanochannelarrays suitable for near-field optical analysis can be modified to havenanoslits. As described above, the nanoslit above the channel is thin topermit sufficient evanescent excitation of the fluorescently-labeledmacromolecules.

In one embodiment of the present invention, nanochannel arrays can beprepared having a sufficiently thin seal thickness suitable fornear-filed optical analysis of fluids in the channels beneath thesealing material. In one embodiment, channels having an opaque sealingmaterial thicker than 100 nm can be modified using a suitablefabrication method to provide a nanoslit in the opaque sealing material.

Suitable fabrication methods for removing material from small areasinclude E-beam lithography and Focus Ion Beam milling. E-beamlithography involves the lithography of ebeam resists followed bydevelopment and reactive ion etching. Focus Ion Beam (FIB) milling ispreferably used, as it requires fewer steps than E-beam lithography. FIBuses a beam of energetic ions such as Gallium ions to sputter materialaway and is capable of resolution down to 20 nm and can etch down manymicrons in principle. FIB is preferred as it enables one to image themilled area immediately after FIB milling the nanoslit structure.

In one embodiment of the present invention, a portion of the sealingmaterial can be deposited inside the channels to form at least one of:an insulating layer, a conducting layer, a hydrophilic layer, ahydrophobic layer, or combinations thereof. In this embodiment, thelayer thickness is typically less than half of the trench width.

In one embodiment, the dimensions, geometry, composition, orcombinations thereof, of the sealing material adjacent to the walls 114can be modified and manipulated for corresponding samples being analyzedin the channels. In a particular embodiment, it is desirable to alterthe surface properties of the sealing material adjacent to the wall 114This is carried out by treating at least some of the channels with asurface-modifying agent to alter the surfaces interior and to saidchannels.

In one embodiment, surface-modifying agents are deposited in thechannels to improve the transport of macromolecules into and through thechannels. Surface-modifying agents are particularly useful where theinternal dimensions (trench depth, trench height, or both) are less thanabout 50 nm. Surface-modifying agents can also reduce or increasehydrophobicity of the surfaces interior to said channels. Nanochannelarrays made according to the present invention can be contacted withsolutions containing surface-modifying agents, such as by submerging thenanochannel array into such solutions. Suitable surface-modifying agentsinclude polyethyleneglycol (PEG), surfactants, Bovin Serum Albumin (BSA)protein solution, surface non-specific binding saturation, andanti-protein sticking agents. Application of a pressure differential,such as vacuum, can be used to assist the treatment of the channels.Application of vacuum is also useful for degassing any fluids inside thechannels.

In certain embodiments of the present invention, the surface-modifyingagent counteracts the electroosmosis effects inside the channels. Whilenot being bound to a particular theory, the electroosmosis effect isusually due to ionized acidic groups immobilized to the matrix (e.g.,attached to the wall) inducing positively charged counter ions in thebuffer that migrate towards the negative electrodes, causing a bulk flowof liquid that migrates in the direction opposite to that of thenegatively charged DNA. Accordingly, reducing electroosmosis effectshelps charged macromolecules to be transported into and along thechannels.

In one embodiment of the present invention, the channels can be at leastsubstantially enclosed on the surface of the substrate and substantiallyopen on the edges of the substrate. As described herein, the channelsare at least substantially enclosed by controlling the deposition of thesealing material. In one embodiment, the channels are substantially openat the edges, which are readily provided by cleaving or cutting thesubstrate to reveal the interior portion of the channels.

In one embodiment, the deposition of the sealing material completelyencloses the plurality of channels. In this embodiment, the sealinglayer is at least as thick as the atoms of the sealing material.Typically, the sealing material surmounting the plurality of channels isless than 500 nanometers thick. In certain embodiments, the sealingmaterial surmounting the plurality of channels can be less than: 100 nm,50 nm, 25 nm, 10 nm, and 5 nm thick. Typically the sealing materialsurmounting the plurality of channels is at least 1 nanometer thick, andmore typically at least 2 nm thick. In certain embodiments of thepresent invention, a step of removing a portion of the sealing materialis used to reduce the thickness of the sealing material above at leastone channel. Sealing material can be removed by a variety of etching andebeam deposition methods as further described herein.

In another aspect of the present invention, there is provided ananofluidic chip that includes a nanochannel array of the presentinvention. Referring to FIG. 5, the nanofluidic chip 200 has ananochannel array 100, a substrate 146, and reservoirs 144 for samplesand waste (or sample collection). Further provided in FIG. 5 areauxiliary sample ports 140 and auxiliary waste ports for handling fluidsample. The reservoirs are in fluid communication with at least one ofthe channels, so that the sample reservoirs are capable of releasing afluid into the channels, and the waste reservoirs are capable ofreceiving a fluid from the channels. Typically the fluids containmacromolecules for analysis.

In certain embodiments of the present invention, the nanofluidic chipcontains at least one sample reservoir is formed in the surface of thesubstrate. Steps to form reservoirs in nanochannel array substrates areprovided above. In this embodiment, at least one waste reservoir influid communication with at least one of the channels. Typically, thenanofluidic chip contains at least 1 sample reservoir. A variety ofother embodiments include at least 96 reservoirs, and even at least 1000reservoirs in the nanofluidic chip.

For use in macromolecular analysis, it is preferred that the nanofluidicchip provides at least a portion of the nanochannel array capable ofbeing imaged with a two-dimensional detector. Imaging of the array isprovided by presenting the sealing material face of the nanochannelarray to suitable apparatus for the collection of emitted signals, suchas optical elements for the collection of light from the nanochannelarray. In this embodiment, the nanofluidic chip is capable oftransporting a plurality of elongated macromolecules from a samplereservoir and across the channels.

In certain embodiments of the present invention, the nanofluidic chipcontains an apparatus for transporting macromolecules from the samplereservoirs, through the channels, and into the waste reservoirs. Asuitable apparatus includes at least one pair of electrodes capable ofapplying an electric field across at least some of the channels in atleast one direction. Electrode metal contacts can be integrated usingstandard integrated circuit fabrication technology to be in contact withat least one sample and at least one collection/waste reservoir toestablish directional electric field. Alternating current (AC), directcurrent (DC), or both types of fields can be applied. The electrodes canbe made of almost any metal, and are typically thin Al/Au metal layersdeposited on defined line paths. Typically at least one end of oneelectrode is in contact with buffer solution in the reservoir.

In certain embodiments of the present invention, the nanofluidic chipcontains at least two pair of electrodes, each providing an electricfield in different directions. In this embodiment, adjacent clusters ofchannels connect individual isolated reservoir. With at least two setsof independent electrodes, field contact can be used to independentlymodulate the direction and amplitudes of the electric fields to movemacromolecules at desired speed or directions.

In another aspect of the present invention, there is provided a system(FIG. 10, 300) that is suitable for carrying out macromolecularanalysis. In the present invention, the system includes a nanofluidicchip as described herein, and an apparatus for detecting at least onesignal transmitted from one or more fluids in the nanochannel array ofthe nanofluidic chip.

In various embodiments of the present invention, the system furtherincludes at least one of the following: a transporting apparatus totransport a fluid through at least one of the channels; a sample loadingapparatus for loading at least one fluid to the sample reservoirs in thenanofluidic chip; and a data processor. The various components of thesystem 300 are connected together, and the general principles ofoperation are illustrated in FIG. 10.

The nanofluidic chip 200 used in the system is typically disposable,individually packaged, and having a sample loading capacity of 1-50,000individual fluid samples. The nanofluidic chip typically has at leastone interconnecting sample delivery microchannel to provide fluidsamples into the channels, as well as sample loading openings and areservoir, or sample loading openings and plates connected with asealing mechanism, such as an O-ring. Metal contacts for connecting theelectrodes 202 and an electric potential generator 216 are also providedin the nanofluidic chips. Suitable metal contacts can be externalcontact patches that can be connected to an externalscanning/imaging/electric-field tuner.

The nanofluidic chip is preferably encased in a suitable housing, suchas plastic, to provide a convenient and commercially-ready cartridge orcassette. Typically the nanofluidic cartridges will have suitablefeatures on or in the housing for inserting, guiding, and aligning thesample loading device with the reservoirs. Insertion slots, tracks, orboth can be provided in the plastic case.

Macromolecular fluid samples that can be analyzed by the system includesfluids from a mammal (e.g., DNA, cells, blood, biopsy tissues),synthetic macromolecules such as polymers, and materials found in nature(e.g., materials derived from plants, animals, and other life forms).Such fluid samples can be managed, loaded, and injected using automatedor manual sample loading apparatus of the present invention.

In one embodiment of the present invention, the system includes anapparatus to excite the macromolecules inside the channels and detectand collect the resulting signals. A suitable apparatus is illustratedin FIG. 10: a laser beam 204 is focused using a focusing lens 206 to aspot on the nanochannel array 100. The generated light signal from themacromolecules inside the channels (not shown) is collected by acollection lens 208, and reflected off a dichroic mirror 218 into anoptical path 220, which is fed into a CCD (charge coupled device)camera. Various optical components and devices can also be used in thesystem to detect optical signals, such as digital cameras, PMTs(photomultiplier tubes), and APDs (Avalanche photodiodes.

In another embodiment of the present invention, the system includes adata processor. The data processor can be used to process the signalsfrom the CCD to project the digital image of the nanochannel array on adisplay 212. The data processor can also analyze the digital image toprovide characterization information, such as macromolecular sizestatistics, histograms, karyotypes, mapping, diagnostics information anddisplay the information in suitable form for data readout 214.

In another aspect of the present invention, there is provided a methodof analyzing at least one macromolecule. In this invention, the analysisincludes the steps of providing a nanofluidic chip according to thepresent invention, providing the at least one sample reservoir with atleast one fluid, said fluid comprising at least one macromolecule;transporting the at least one macromolecule into the at least onechannel to elongate said at least one macromolecule; detecting at leastone signal transmitted from the at least one elongated macromolecule;and correlating the detected signal to at least one property of the atleast one macromolecule.

In one embodiment of the present invention, the method of analyzing amacromolecule includes wetting the channels using capillary action witha buffer solution or a buffer solution containing macromolecules.Macromolecules such as polymers and DNA can introduced into nanochannelarrays by electric field.

Various macromolecules can be analyzed using the present method. Foranalyzing DNA typical process conditions include providing dilutesolutions of DNA which are stained at a ratio of 4:1 to 10:1 basepair/dye with a suitable dye. Suitable dye stains include TOTO-1,BOBO-1, BOBO-3 (Molecular Probes, Eugene, Oreg.). Solutions of stainedDNA can be further diluted and treated with an anti-oxidant and ananti-sticking agent.

In one embodiment of the present invention, the method of analyzing amacromolecule includes the sizing of one DNA. One DNA macromolecule canbe extracted from a single cell or spore, such as anthrax, and suitablytransported (e.g., in a polymerized gel) to avoid breakage.

Macromolecular fluid samples can be loaded through reservoirs in thenanofluidics chip and transported via interconnecting microchannels. Themacromolecules are partially elongated before one end of themacromolecule enters the channels; they are substantially fullyelongated when completely inside the channels. The fluorescent signalscan be excited by the appropriate excitation sources and emissionsignals can be collected via imaging camera or detectors, in a linearscanning mode or CCD image integration. The signals collected can beanalyzed by data processing software and user-defined major parameters(intensity/photons, major axis, minor axis, background signal) can berecorded and measured.

The length of a single DNA can be detected/reported and intensityprofile can be plotted. In various embodiments of the present invention,the method of analyzing a macromolecule includes correlating thedetected signal to at least one of the following properties: length,conformation, and chemical composition. Various macromolecules that canbe analyzed this way include, biopolymers such as a protein, apolypeptide, and a nucleic acid such as RNA or DNA. For DNA nucleicacids, the detected signals can be correlated to the base pair sequenceof said DNA.

The typical concentration of the macromolecules in the fluid will be onemacromolecule, or about at least attogram per ml, more typically atleast one femtogram per ml, more typically at least one picogram per ml,and even more typically at least one nanogram per ml. Concentrationswill typically be less than 5 micrograms per milliliter and moretypically less than 0.5 micrograms per milliliter.

In one embodiment of the present invention, the method of analyzing amacromolecule measures the length of macromolecules having an elongatedlength of greater than 150 nanometers, and typically greater than 500nanometers, 1 micron, 10 microns, 100 microns, 1 mm, 1 cm, and 10 cmlong.

DNA having greater than 10 base pairs can also be analyzed using thepresent methods. Typically, the number of base pairs measured can begreater than 100 base pairs, greater than 1,000 base pairs, greater than10,000 base pairs, greater than 20,000 base pairs, greater than 40,000base pairs, and greater than 80,000 base pairs. DNA having more than 1million, 10 million, and even 100 million basepairs can be analyzed withthe present methods.

In one embodiment of the present invention, the methods can be used toanalyze one or more of the following: restriction fragment lengthpolymorphism, a chromosome, and single nucleotide polymorphism.

The following abbreviations are used herein: “nm” is nanometer, “mTorr”is milli Torr.

General Procedures

After NIL and etching, non-uniform deposition of sealing material wasprovided by e-beam evaporation with a tilted sample wafer at variousangles or sputter deposition using a large source target. This step wasused to both reduce the trench width and seal the channels.

Generally, 100-340 nm of SiO₂ was deposited onto the patternedsubstrate. Effective sealing was achieved with various depositionconditions that were tested. At gas pressure of 30 mTorr, RF power of˜900 W, and DC bias of 1400 V, a deposition rate of ˜9 nm/min wasachieved. At lower pressure of 5 mTorr, the deposition rate wasincreased to an estimated 17 nm/min. Sealing material was deposited onthe patterned substrate by sputtering at 5 mTorr.

EXAMPLES

In the following examples, nanochannel arrays were prepared using aprocess to deposit SiO₂ sealing material on patterned substrates bysputtering. Channel openings were prepared by cleaving the substrate andimaged by Scanning Electronic Microscope (SEM). Results are as followsand shows that the trench widths are narrowed by the deposition of thesealing material using sputtering:

Example 1

A 100 mm silicon substrate was provided having a plurality of parallellinear channels that had an 85 nm trench width and a 200 nm trenchheight (FIG. 7a ). This substrate was sputtered at a gas pressure of 5mTorr according to the general procedures given above. After sputtering,the channels had a 52 nm trench width, a 186 nm trench height, and aseal thickness of 200 nm (FIG. 7b ). Apparently, the trench heightincreased slightly as a result of the sealing process forming acone-shaped seal above the channel.

Example 2

A patterned substrate having a 65 nm trench width and about 100 nmtrench height prior to sputtering formed a nanochannel array having a 17nm trench width, a 68 nm trench height, and a channel seal thickness ofabout 350 nm. Sputtering gas pressure was 5 mTorr.

Example 3

A patterned substrate having a 50 nm trench width and about 80 nm trenchdepth prior to sputtering formed a nanochannel array having a 10 nmtrench width, 51 nm trench height, and a channel seal thickness of 350nm. Sputtering gas pressure was 5 mTorr.

Example 4

A substrate containing a two-dimensional array of pillars was made usinga two-step NIL process with the channel mold rotated 90° between theimprinting steps (FIG. 6a ). The pillar array structure is subsequentlycompletely sealed with silicon dioxide using a 29 minute depositiontime. The seal thickness was about 500 nm. A profile view of the channelis depicted in FIG. 6b and a top view of the completely sealednanochannel array is depicted in FIG. 6 d.

Example 5

Example 4 was repeated except the sputter deposition time was 17 minutesto provide a nanochannel array that is not completely sealed. A top viewscanning electron micrograph of this nanochannel array is provided inFIG. 6d . The seal thickness was 300 nm.

Example 6

A nanoslit is provided in a channel prepared with a silicon dioxidesealing material for carrying out near-field analysis having a sealthickness greater than about 100 nm is modified by using FIB to create ananoslit having a thickness less than 100 nm. FIG. 8 shows a schematicof how the deposited sealing material on a nanochannel array is firstmilled away using FIB from the sealing material situated above thesealed channels. Subsequently, aluminum is deposited to create an opaquelayer to provide optical contrast at the slit.

Example 7

This example shows how a nanochannel array can be prepared from asubstrate having a plurality channels larger than 150 nm wide by 150 nmdeep. A substrate is prepared by photolithography techniques to providea plurality of channels with width of greater than 1.5 micron usingconventional optical lithography techniques: Contact aligner such asKarl Suss MA-6 to provide a pattern resolution at low micron level;Industrial projection stepper. The angle of the incident depositing beamof sealing material is varied to reduce the trench width and height toless than 150 nm and 150 nm, respectively, and to substantially seal byproviding shallow tangential deposition angles.

Example 8

This example provides a nanochannel array using an e-beam technique. Asubstrate is provided as in Example 1. Silicon dioxide is deposited byan e-beam (thermo) evaporator (Temescal BJD-1800) onto the substrate.The substrate is placed at various angles incident to the depositingbeam from the silicon dioxide source target; the deposition rate is setto about 3 nm/minute and 150 nm of sealing material is deposited inabout 50 minutes.

Example 9

In this example, a nanochannel array is contacted with asurface-modifying agent. A nanochannel array made according to Example 1is submerged in a surface-modifying agents solutions containingpolyethylene glycol inside a vacuum chamber overnight to facilitatewetting and treatment of the channels and degas the air bubbles thatmight be trapped inside the channels.

Example 10

This example shows the preparation of a nanochannel array having a metalsealing material. An e-beam (thermo) evaporator (Temescal BJD-1800) wasused to deposit Chromium (Cr) onto a nanochannel array chip (trenchwidth 80 nm, trench depth 80 nm, SiO2/Si substrate). The substrate wasplaced at various angles to the incident depositing beam from the sourcetarget, the deposition rate was set at 2.0-3.6 nm/minute. The resultingtrench width was 20 nm, trench depth less than 80 nm, and the channelswere substantially closed.

Example 11

This example shows the process of adding an optically opaque layer to ananochannel array. A nanochannel array made according to Example 3 isplaced perpendicular to the incident depositing beam to provide anopaque layer less than 50 nm thick. An aluminum source target isselected for depositing on top of the SiO2 sealing material above thesealed channels. The deposition rate was set at 2.0˜3.6 nm/minute.

Example 12

This example describes the steps needed to provide a near-field slit ina nanochannel array. FIB was used to mill narrow slits less than 50 nmin width in the direction perpendicular to the long axis of thenanochannel array of Example 11. The depth of the FIB milling wascontrolled to expose the underlying thin SiO2 sealing material above thenanochannel array.

Example 13

This example describes how to provide a sample reservoir with ananochannel array to provide a nanofluidic chip. A nanochannel arrayhaving 100 nm wide, 100 nm deep channels was made according to generalprocedures of Example 1. The nanochannel array was spin-coated with aphotoresist and imaged with a photomask to provide regions on oppositeends of the nanochannel array. The exposed areas were etched usingreactive ion etching to expose the channel ends and to provide amicron-deep reservoir about a millimeter wide on the opposite ends ofthe channels at the edge of the substrate.

Example 14

This example describes how to fill a nanofluidic chip with a fluidcontaining DNA macromolecules to analyze the DNA. A cylindrical-shapedplastic sample-delivery tube of 2 mm diameter was placed in fluidcommunication with one of the reservoirs of the nanochannel array ofExample 13. The delivery tube is connected to an external sampledelivery/collection device, which is in turn connected to apressure/vacuum generating apparatus. The channels are wetted usingcapillary action with a buffer solution. A buffer solution containingstained lambda phage macromolecules (lambda DNA) were introduced intothe nanochannel array by electric field (at 1-50 V/cm); the solutionconcentration was 5 microgram/mL and the lambda DNA was stained at aratio of 10:1 base pair/dye with the dye TOTO-1 (Molecular Probes,Eugene, Oreg.). This solution of stained DNA was diluted to 0.1-0.5microgram/mL into 0.5×TBE (tris-boroacetate buffer at pH 7.0) containing0.1M of an anti-oxidant and 0.1% of a linear polyacrylamide used as ananti-sticking agent.

Example 14

A nanofluidic chip made according to Example 12, having channeldimensions of 100 nm×100 nm was filled using capillary action with abuffer solution containing stained genomic DNA to draw the DNAmacromolecules into the channels with an electric field. Bacteria phageDNA molecules Lambda (48.5 kb) and T4 (168.9 kb) were stained with thedye TOTO-1 and BOBO-3 respectively. This solution of stained DNA wasdiluted to 0.5 μg/mL into 0.5×TBE containing 0.1M dithiothreatol as ananti-oxidant and 0.1% of a linear acrylamide used as an anti-stickingagent). A Nikon Eclipse TE-300 inverted microscope with a 60× (N.A. 1.4)oil immersion objective was used with an Ar:K laser (Coherent Lasers) asan excitation source at 488 nm and 570 nm. A Roper Scientific Pentamaxintensified cooled CCD camera with a 512×512 pixel array and 16 bitsdigital output was used to image the molecules. Digital image wasanalyzed using a data processor by NIH Image software. FIG. 9b shows anintegrated image of the stretched Lambda and T4 phage genomes side byside in the channels. The inset of 9 b shows the near perfect linear fitof the directly measured length obtained from the digital image plottedagainst their genome size, (R2 is 0.99996). FIG. 9c shows an array offluorescently-labeled genomic DNA molecules aligned and stretched in thechannels with the size ranging from 10 kb to 194 kb. This shows thatmillions of center-meter long parallel channels could be fabricated overthe whole wafer. Accordingly, the entire length of genomic DNA moleculescan be stretched and analyzed.

Example 15

Example 14 is repeated, but with a 96 multiple reservoir system. Ananofluidic chip made according to Example 12 is modified with aphotomask to provide 96 sample reservoirs, each reservoir connected to1000 channels along one edge of the 100 mm substrate. 96 different DNAsamples are delivered and injected using capillary fibers connected tothe sample reservoirs. 96 collection reservoirs are connected to thecorresponding ends of the channels to collect the DNA samples.

Example 16

This example describes a system used for carrying out analysis ofmacromolecules. The system contains an automated 96-capillaryauto-injection sample loader to deliver 96 macromolecular fluid samplesinto the delivery ports of a nanofluidic cartridge. The nanofluidiccartridge is a nanofluidic chip encased by a plastic polycarbonatehousing, having delivery ports and collection ports for connection tomicrocapillary tubing, and embedded metal contacts for connection toelectrodes on the nanofluidic chip. The cartridge can be inserted in acartridge holder, which is integrated with an a laser excitation sourceand suitable optical components to provide the excitation of andcollection of optical signals emanating from sample fluids within thenanochannel arrays of the nanofluidic chip. The signaldetection/collection apparatus is a cooled CCD digital camera. Signalsfrom the digital camera are analyzed by a data processor using NIH imageanalysis software, and displayed on a monitor.

Example 17

This example describes how to use the system of Example 16 to size oneDNA macromolecule. A single Anthrax spore is lysed to extract its entiregenomic contents (DNA) with 10 microliters of a buffer solution andstained with fluorescent dyes. The sample loader is inserted into thedelivery ports of the cartridge and injects the DNA-containing fluid.The electrodes are activated and the DNA macromolecules are transportedinto the nanochannel array, where they become elongated. The fluorescentstains on the DNA are excited by the excitation source, and theiremission signals are collected using the CCD camera. The signalscollected, analyzed and recorded for intensity and position by the dataprocessor. The length of a single DNA is detected and intensity profileis plotted.

In another aspect of the present invention, there is provided 144. Acartridge comprising at least one nanofluidic chip, said cartridgecapable of being inserted and removed from a system for carrying outmacromolecular analysis, said at least one nanofluidic chip comprisingat least one nanonanochannel array, said nanonanochannel arraycomprising

a surface having a plurality of channels in the material of the surface,said channels having a trench width of less than about 150 nanometersand a trench depth of less than 200 nanometers;

at least some of the channels being surmounted by sealing material torender such channels at least substantially enclosed.

The invention claimed is:
 1. A nanofluidic chip, comprising: a)nanochannel array, comprising: a substrate having a surface; at leastone channel in the surface, wherein the at least one channel has atrench width of less than 100 nanometers and a trench depth of less than100 nanometers; and the at least one channel is capable of admitting afluid; b) at least one sample reservoir in fluid communication with theat least one channel, said sample reservoir capable of releasing afluid; and c) at least one waste reservoir in fluid communication withthe at least one channel, said waste reservoir capable of receiving afluid.
 2. The nanofluidic chip of claim 1, wherein the at least onechannel is one of a plurality of parallel channels in the surface. 3.The nanofluidic chip of claim 1, wherein the at least one channel issurmounted by sealing material to render the channel at leastsubstantially enclosed.
 4. The nanofluidic chip of claim 1 wherein theat least one sample reservoir is formed in the surface of the substrate.5. The nanofluidic chip of claim 1 further comprising at least one wastereservoir in fluid communication with the at least one channel.
 6. Thenanofluidic chip of claim 1 capable of transporting a plurality ofelongated macromolecules from a sample reservoir and across the at leastone channel.
 7. The nanofluidic chip of claim 1 further comprising atleast one pair of electrodes capable of applying an electric fieldacross the at least one channel in at least one direction.
 8. Thenanofluidic chip of claim 7 wherein at least two pair of electrodes eachprovides an electric field in different directions.
 9. A system,comprising: A) a nanofluidic chip, comprising: a) nanochannel array,comprising: a substrate having a surface; and at least one channel inthe surface capable of admitting a fluid, wherein the at least onechannel has a trench width of less than 100 nanometers and a trenchdepth of less than 100 nanometers; and b) at least one sample reservoirin fluid communication with the at least one channel, said samplereservoir capable of releasing a fluid; and B) an apparatus fordetecting at least one signal transmitted from the at least one fluid inthe nanochannel array.
 10. The nanofluidic chip of claim 9, wherein theat least one channel is one of a plurality of parallel channels in thesurface.
 11. The nanofluidic chip of claim 9, wherein the at least onechannel is surmounted by sealing material to render the channel at leastsubstantially enclosed.
 12. The system according to claim 9, furthercomprising a transporting apparatus to transport a fluid through at theat least one channel.
 13. The system according to claim 9, furthercomprising a sample loading apparatus for loading at least one fluid tothe at least one sample reservoir.
 14. The system according to claim 9,further comprising a data processor.
 15. A method of analyzing at leastone macromolecule, comprising the steps of: providing a nanofluidicchip, comprising: a) nanochannel array, comprising: a substrate having asurface; at least one nanochannel in the surface, wherein the at leastone channel has a trench width of less than 100 nanometers and a trenchdepth of less than 100 nanometers; and the at least one channel iscapable of admitting a fluid; b) at least one sample reservoir in fluidcommunication with the at least one channel, said sample reservoircapable of releasing a fluid; and c) at least one waste reservoir influid communication with the at least one channel, said waste reservoircapable of receiving a fluid; transporting at least one macromolecule ina fluid sample into the at least one nanochannel, such that the at leastone macromolecule is in an elongated state within the at least onenanochannel; maintaining the at least one macromolecule in an elongatedstate within a region of the at least one nanochannel having asubstantially constant width; detecting at least one signal from the atleast one elongated macromolecule within the at least one nanochannel;and correlating the detected signal to at least one property of the atleast one macromolecule.
 16. The method of claim 15, wherein the atleast one macromolecule is transported into the nanochannel by applyingan electric current.